CN104012416A - Method for efficiently regenerating Sapium sebiferum leaf disc - Google Patents
Method for efficiently regenerating Sapium sebiferum leaf disc Download PDFInfo
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- CN104012416A CN104012416A CN201410300596.7A CN201410300596A CN104012416A CN 104012416 A CN104012416 A CN 104012416A CN 201410300596 A CN201410300596 A CN 201410300596A CN 104012416 A CN104012416 A CN 104012416A
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Abstract
The invention discloses a method for efficiently regenerating a Sapium sebiferum leaf disc. The method comprises the following steps: cutting leaves of aseptic Sapium sebiferum seedlings into leaf discs, and conducting induction culturing on the leaf discs through a cluster bud induction culture medium to form cluster buds; conducting micropropagation and elongation on the cluster buds in a micropropagation and elongation culture medium; and after the buds grow to 2-3cm, transferring into a rooting culture medium for rooting culturing, thus obtaining strong Sapium sebiferum test-tube plantlets after 25-30 days. Compared with the prior art, the method has the following advantages that raw materials are easily available and abundant, the regeneration efficiency is high, the culture period is short, hormone components added to the culture mediums are simple, the operation is convenient, and the like, and the method can realize fast propagation of excellent wild single plants, and keep the germplasm characteristics of a female parent, thus realizing the breeding of variety, laying a foundation for the genetic transformation of Sapium sebiferum, being beneficial to quickening the genetic improvement of Sapium sebiferum, and providing technical support for the fast propagation of excellent anaphase Sapium sebiferum plant systems in the later period.
Description
Technical field
The present invention relates to biological technical field, the method for in particular a kind of Chinese tallowtree leaf dish highly efficient regeneration.
Background technology
Chinese tallow tree (Sapium sebiferum) is the perennial deciduous tree of Euphorbiaceae sapium.Chinese tallow tree is stronger to the adaptability of soil, in great soil groups such as red soil, yellow earth, yellowish-brown soil, purple soil, brown earth, and at the soil from sand to sticky differing texture, and acid, neutral or subalkaline soil, all can grow, and be one of arbor species that salt-resistance is strong.Chinese tallow tree requires there is higher soil moisture, and ability short-term ponding.There are certain wind resistance and more resistance to dry morning barren simultaneously.In addition, Chinese tallow tree has stronger resistance to poisonous hydrogen fluoride gas.Chinese tallow tree is distributed widely in China subtropical and tropical zones, is the peculiar economic tree of China.Chinese tallow tree is multipurpose seeds, and seed is outer by wax, can be used for making the industrial raw materials such as soap, candle and paint; Seed oil yield is up to 53%, and the liquid oil that kind benevolence is squeezed can be used as the raw material of biodiesel.Chinese tallow tree is listed in one of " China's four large woody oil tree species "; Autumn leaves is scarlet, purple or foresythia, also can do courtyard greening and street tree.No matter Chinese tallow tree is to use economic crops as oil plant, or as viewing and admiring green tree species, all caused extensive concern in recent years.
But the virgin phase of the ubiquity of Chinese tallow tree plantation is at present long, fruit yield is low, lack the problems such as improved seeds, has greatly limited it and has cultivated on a large scale and commercial application as energy-source plant.Therefore, strengthen its breed improvement, cultivate short virgin phase, high yield, high-quality and pest-resistant Chinese tallow tree improved seeds are the most urgent tasks of current development Chinese tallow tree Biodiesel.About the Study on Genetic Basis weakness of Chinese tallow tree, it is blank that breeding work still belongs at present; Simultaneously, because Chinese tallow tree is as perennial woody plant, its breed of variety ubiquity generation cycle is long, the problem such as heterozygosis highly on genetics, and conventional breeding is difficult to meet breeding demand.Breeding process can be greatly accelerated to a certain extent taking transgenic technology as basic molecular breeding means, but the tissue culture regeneration system of efficient stable need to be depended on.The numerous support that also needs tissue culture expanding propagation technology of expansion of elite plant strain (kind) simultaneously.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the present invention uses animal nutrition, and the method for a kind of Chinese tallowtree leaf dish highly efficient regeneration is provided, and for breed improvement and the Fast-propagation of realizing Chinese tallow tree lay the foundation, advances the development of Chinese tallow tree Biodiesel.
The present invention is achieved by the following technical solutions:
A method for Chinese tallowtree leaf dish highly efficient regeneration, comprises the steps:
(1) get the blade of Chinese tallow tree aseptic seedling, be cut into multiple 0.3 × 0.5cm
2the leaf dish of size;
(2) multiple leaf dishes are inoculated on inducing clumping bud medium and are cultivated, after 21～28d, induction forms Multiple Buds;
(3) again Multiple Buds is seeded in in corresponding propagation and elongation medium, to carry out the expansion of bud numerous and extend;
(4) grow to 2～3cm when high until bud, proceed to root media and carry out culture of rootage, after 25～30d, obtain healthy and strong Chinese tallow tree test-tube plantlet;
Wherein, the minimal medium of described inducing clumping bud medium, propagation and elongation medium, these three kinds of medium of root media is the MS medium that contains 30g/L sucrose and 7.0g/L agar, and described inducing clumping bud medium also includes one or both plant hormones.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, in described step (1), the have drawn from blade of the Chinese tallow tree aseptic seedling full extension obtaining by field Chinese tallow tree plants stems Duan Zupei 30～50d of described blade.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, in described step (2), the vaccination ways of described leaf dish on inducing clumping bud medium be leaf dish proximal ends upward.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, in described step (2), described inducing clumping bud medium is: MS+0.01～3.0mg/L TDZ or 0.1～2.0mg/L6-BA+0.0～2.0mg/L NAA+30g/L sucrose+7.0g/L agar.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, in described step (3), described propagation and elongation medium are: MS+0.3～1.5mg/L6-BA+0.0～0.2mg/L NAA+30g/L sucrose+7.0g/L agar.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, in described step (4), described root media is: 1/2MS+0.1～3.0mg/L IBA+30g/L sucrose+7.0g/L agar.
As the preferred embodiment of the method for above-mentioned a kind of Chinese tallowtree leaf dish highly efficient regeneration, described regeneration condition of culture is: 25 ± 2 DEG C of temperature, and intensity of illumination 2000～2500lx, light application time is 13～14h.
The present invention has the following advantages compared to existing technology:
The method of a kind of Chinese tallowtree leaf dish provided by the present invention highly efficient regeneration, adopt the mode of artificial induction's Multiple Buds regeneration, choosing the leaf dish that the blade of Chinese tallow tree aseptic seedling is cut into is material, pass through hormone regulating and controlling, under isolated culture condition, cultivate regeneration, there is following outstanding advantages: one, blade has drawn from by the blade of the Chinese tallow tree aseptic seedling full extension of field Chinese tallow tree plants stems Duan Zupei acquisition, can keep maternal germplasm feature, draw materials conveniently, material is abundant, can realize numerous soon to field fine individual plant, realize breed of variety; Its two, Multiple Buds reproduction speed is fast, leaf dish only needs the induction through 3～4 weeks, renewable go out a large amount of Multiple Buds, the Multiple Buds bearing again through the each leaf dish of test statistics is 10-30; Its three, regeneration efficiency is high, inductivity reaches 100%; Its four, the cycle that the regeneration of whole leaf dish is cultivated is only 75-80d, cultivation cycle is short; Its five, the expansion of bud is numerous and extend and only need on a medium, carry out, step is simplified, simple to operate; Its six, in regenerative process, medium used has only added one or both plant hormones, adds hormone composition simple, cost-saving, handled easily.Therefore, the method for Chinese tallowtree leaf dish provided by the invention highly efficient regeneration, for Chinese tallow tree genetic transformation provides support, is conducive to accelerate the process of Chinese tallow tree genetic improvement; For the later stage, the Fast-propagation of Chinese tallow tree elite plant strain provides technical support.
Brief description of the drawings
Fig. 1 is the schematic diagram that blade is cut into Ye Panhou.
Fig. 2 is the Multiple Buds schematic diagram that leaf dish forms after the induction of inducing clumping bud medium is cultivated.
Fig. 3 be Multiple Buds through propagation and elongation medium carry out the expansion of bud numerous and extend after structural representation.
Fig. 4 is the schematic diagram of Multiple Buds after root media carries out culture of rootage.
Fig. 5 is that Chinese tallow tree test-tube plantlet carries out hardening, domestication schematic diagram.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The method of a kind of Chinese tallowtree leaf dish highly efficient regeneration that the present embodiment provides, comprises the steps:
(1) get the blade of Chinese tallow tree aseptic seedling, be cut into multiple 0.3 × 0.5cm
2the leaf dish of size, as shown in Figure 1; Wherein, the blade blade of Chinese tallow tree aseptic seedling full extension obtaining by field Chinese tallow tree plants stems Duan Zupei 30～50d that preferably has drawn from;
(2) multiple leaf dishes are inoculated on inducing clumping bud medium and are cultivated, the vaccination ways of leaf dish on inducing clumping bud medium be leaf dish proximal ends upward, after 21～28d induction form Multiple Buds; As shown in Figure 2;
(3) again Multiple Buds is seeded in in corresponding propagation and elongation medium, to carry out the expansion of bud numerous and extend, as shown in Figure 3;
(4) grow to 2～3cm when high until bud, proceed to root media and carry out culture of rootage, obtain healthy and strong Chinese tallow tree test-tube plantlet after 25～30d, rooting rate is up to 96.3%, as shown in Figure 4; Finally Chinese tallow tree test-tube plantlet is carried out to hardening, domestication, can obtain healthy and strong Chinese tallow tree regeneration plant, as shown in Figure 5.
Wherein, whole regeneration condition of culture is: 25 ± 2 DEG C of temperature, and intensity of illumination 2000～2500lx, light application time is 13～14h.The minimal medium of inducing clumping bud medium, propagation and elongation medium, these three kinds of medium of root media is the MS medium that contains 30g/L sucrose and 7.0g/L agar.
In step (2), inducing clumping bud medium is: MS+0.01～3.0mg/L TDZ or 0.1～2.0mg/L 6-BA+0.0～2.0mg/L NAA+30g/L sucrose+7.0g/L agar.
In step (3), propagation with elongation medium is: MS+0.3～1.5mg/L 6-BA+0.0～0.2mg/L NAA+30g/L sucrose+7.0g/L agar.
In step (4), root media is: 1/2MS+0.1～3.0mg/L IBA+30g/L sucrose+7.0g/L agar.
Through verification experimental verification, the cycle that whole leaf dish regeneration is cultivated is only 75-80d, and cultivation cycle is short.Regeneration efficiency is high, and inductivity reaches 100%.Method provided by the present invention has the facility of drawing materials, material is abundant, regeneration efficiency is high, cultivation cycle is short, it is simple that medium adds hormone composition, the advantage such as easy to operate, and the method that provides of this patent can realize numerous soon to field fine individual plant, can keep maternal germplasm feature, realize breed of variety; For Chinese tallow tree genetic transformation provides support, be conducive to accelerate the process of Chinese tallow tree genetic improvement, for the later stage, the Fast-propagation of Chinese tallow tree elite plant strain provides technical support.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (7)
1. a method for Chinese tallowtree leaf dish highly efficient regeneration, is characterized in that, comprises the steps:
(1) get the blade of Chinese tallow tree aseptic seedling, be cut into multiple 0.3 × 0.5cm
2the leaf dish of size;
(2) multiple leaf dishes are inoculated on inducing clumping bud medium and are cultivated, after 21～28d, induction forms Multiple Buds;
(3) again Multiple Buds is seeded in in corresponding propagation and elongation medium, to carry out the expansion of bud numerous and extend;
(4) grow to 2～3cm when high until bud, proceed to root media and carry out culture of rootage, after 25～30d, obtain healthy and strong Chinese tallow tree test-tube plantlet;
Wherein, the minimal medium of described inducing clumping bud medium, propagation and elongation medium, these three kinds of medium of root media is the MS medium that contains 30g/L sucrose and 7.0g/L agar, and described inducing clumping bud medium also includes one or both plant hormones.
2. the method for a kind of Chinese tallowtree leaf dish as claimed in claim 1 highly efficient regeneration, is characterized in that, in described step (1), described blade has drawn from by the blade of the Chinese tallow tree aseptic seedling full extension of field Chinese tallow tree plants stems Duan Zupei 30～50d acquisition.
3. the method for a kind of Chinese tallowtree leaf dish as claimed in claim 1 highly efficient regeneration, is characterized in that, in described step (2), the vaccination ways of described leaf dish on inducing clumping bud medium be leaf dish proximal ends upward.
4. the method for a kind of Chinese tallowtree leaf dish as claimed in claim 1 highly efficient regeneration, it is characterized in that, in described step (2), described inducing clumping bud medium is: MS+0.01～3.0mg/L TDZ or 0.1～2.0mg/L6-BA+0.0～2.0mg/L NAA+30g/L sucrose+7.0g/L agar.
5. the method for a kind of Chinese tallowtree leaf dish as claimed in claim 1 highly efficient regeneration, it is characterized in that, in described step (3), described propagation and elongation medium are: MS+0.3～1.5mg/L6-BA+0.0～0.2mg/L NAA+30g/L sucrose+7.0g/L agar.
6. the method for a kind of Chinese tallowtree leaf dish as claimed in claim 1 highly efficient regeneration, is characterized in that, in described step (4), described root media is: 1/2MS+0.1～3.0mg/L IBA+30g/L sucrose+7.0g/L agar.
7. the method for a kind of Chinese tallowtree leaf dish highly efficient regeneration as described in as arbitrary in claim 1 to 6, is characterized in that, described regeneration condition of culture is: 25 ± 2 DEG C of temperature, and intensity of illumination 2000～2500lx, light application time is 13～14h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957041A (en) * | 2015-07-16 | 2015-10-07 | 中国科学院合肥物质科学研究院 | Method for inducing regeneration of triadica sebifera plant by utilizing leaf stalk as explant |
| CN104982337A (en) * | 2015-07-28 | 2015-10-21 | 中国科学院合肥物质科学研究院 | Method for improving regeneration efficiency of wild tallow excellent strain stem |
| CN105766634A (en) * | 2016-03-10 | 2016-07-20 | 中国科学院合肥物质科学研究院 | In vitro regeneration method of adventitious roots of sapium sebiferum roxb |
| CN106489742A (en) * | 2016-12-06 | 2017-03-15 | 中国科学院合肥物质科学研究院 | A kind of method that Cortex Sapii Radiciss regeneration plant is obtained by Anther Culture |
| CN109076961A (en) * | 2018-10-29 | 2018-12-25 | 中国科学院合肥物质科学研究院 | A kind of method of tallow tree tissue culture adventitious bud outside sprout-cultivating-bottle nursery |
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| CN101057556A (en) * | 2007-05-23 | 2007-10-24 | 江苏省农业科学院 | Method for high frequency plant regenerating of tallow tree tissue culture adventitious bud |
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| M. KOTWAL MRIDULA, ET AL.: "Rapid multiplication of Sapium sebiferum Roxb. by tissue culture", 《PLANT CELL TISSUE ORGAN CULTURE》, vol. 2, 31 December 1983 (1983-12-31), pages 133 - 139 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957041A (en) * | 2015-07-16 | 2015-10-07 | 中国科学院合肥物质科学研究院 | Method for inducing regeneration of triadica sebifera plant by utilizing leaf stalk as explant |
| CN104982337A (en) * | 2015-07-28 | 2015-10-21 | 中国科学院合肥物质科学研究院 | Method for improving regeneration efficiency of wild tallow excellent strain stem |
| CN104982337B (en) * | 2015-07-28 | 2017-12-01 | 中国科学院合肥物质科学研究院 | A kind of method for improving field Chinese tallow tree elite plant strain leaf regeneration efficiency |
| CN105766634A (en) * | 2016-03-10 | 2016-07-20 | 中国科学院合肥物质科学研究院 | In vitro regeneration method of adventitious roots of sapium sebiferum roxb |
| CN106489742A (en) * | 2016-12-06 | 2017-03-15 | 中国科学院合肥物质科学研究院 | A kind of method that Cortex Sapii Radiciss regeneration plant is obtained by Anther Culture |
| CN109076961A (en) * | 2018-10-29 | 2018-12-25 | 中国科学院合肥物质科学研究院 | A kind of method of tallow tree tissue culture adventitious bud outside sprout-cultivating-bottle nursery |
| CN109076961B (en) * | 2018-10-29 | 2022-02-11 | 中国科学院合肥物质科学研究院 | Method for tissue culture adventitious bud ex-vitro rooting seedling culture of tallow trees |
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Application publication date: 20140903 |